Apart from the Cas9-guide vector, I'm using a donor vector containing left and right homologous arms and a puromycin resistance casette. After antibiotic selection, resistant cells grow as isolated clones which I can pick up and analize separately by Western blot. My problem here is, even if they survive puromycin addition, most of those clones still express nearly 100% of the protein. I don't understand how is this possible as, in theory, the insertion of the casette that provides puro resistance should be enough to guarantee the gene has been disrupted. I've considered the possibility that only one allele is being knocked down (thus explaining why cells are resistant to puromycin) and, as a response, the other one is over-producing the protein in some kind of compensation mechanism, resulting in no visible silencing at the protein level.
Any thoughs on how could I improve the knocking out efficieny (in both alleles) and/or the positive selection of completely silenced clones?
Thank you in advance,
Isabel.
have a look at (we are actually using this optimised vector also, it works well)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918502/
Previous work has indicated that the sgRNA expression level limits CRISPR/Cas9 function in human cells (Jinek et al., 2013). Indeed, the observed nucleolus-like signal possibly came from dCas9 proteins that were not bound to sgRNA. Therefore, we modified the sgRNA design to increase its stability and to enhance its assembly with the dCas9 protein (Figures 1C and S1C). To avoid premature termination of U6 Pol-III transcription, we removed a putative Pol-III terminator (4 consecutive U’s) in the sgRNA stem-loop by an A-U basepair flip (Figures 1C, sgRNA(F)) (Nielsen et al., 2013). To improve sgRNA-dCas9 assembly, we extended the dCas9-binding hairpin structure (Figures 1C, sgRNA(E)).
see also
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699467/
Hey Isabel,
is the knock out of the gene lethal - therefore the compensation effect? And I am not sure if i got your problem right. You integrated the puro cassette via HDR and thereby replaced your GOI? If you want a complete knock down you could take these cells and try another disruption of the CDS of the other allel with your puro resistant cells .
Hi Jan,
Thank you very much for your answer.
The knock out of my gene is not lethal; actually, I obtained a completely silenced clone once following this protocol. But I need more of them and in more cell lines, and that is why I'm looking for a way to improve the efficiency of this method.
To my knowledge, the Cas9 nuclease is supposed to be targeted to my GOI and to disrupt it, causing a double strand break (DSB). Furthermore, there is the puro casette (provided in a different plasmid) whose homologous arms should allow its insertion in the GOI when the DSB is repaired via homologous recombination. This is a low percentage of the cases, but still I was expecting at least some of the puro resistant clones to be silenced at the protein level. However, I don't see that: all the resistant clones show no changes regarding protein expression when compared to the control.
I hope I explained myself well :)
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